title: Cryoprotectant-free ultra-rapid freezing of human spermatozoa in cryogenic vials.
abstract: The objective of this study was to investigate the effect of ultra-rapid freezing (direct immersion in liquid nitrogen) on human spermatozoa in cryogenic vials (≥0.5 ml) at different concentrations of sucrose. After swim-up, the sperm suspensions (N = 58) were diluted with sperm preparation medium and divided into six aliquots: swim-up (fresh), conventional freezing group (slow freezing) and four ultra-rapid freezing groups containing sucrose at different concentrations (0.15 m, 0.20 m, 0.25 m and 0.30 m). Sperm motility, progressive motility, plasma membrane integrity, DNA stability and acrosome integrity of fresh and cooled-warmed spermatozoa were analysed. The progressive motility, plasma membrane and acrosome integrity of spermatozoa in the 0.20 m sucrose group were significantly higher than those of the slow freezing group (47.5 ± 6.8% versus 36.4 ± 8.7%, 73.2 ± 6.9% versus 63.9 ± 6.3%, 53.7 ± 10.0% versus 35.9 ± 9.7% respectively, P < 0.05). However, no differences were found in sperm motility or DNA stability (58.5 ± 6.3% versus 54.2 ± 5.3%, 90.1 ± 2.8% versus 87.2 ± 4.7%, P > 0.05 respectively) between the 0.20 m sucrose and the slow freezing group. No differences were found between the ultra-rapid and slow freezing group at the other concentrations of sucrose. Our findings suggest that the method of ultra-rapid freezing of human spermatozoa in cryogenic vials with a solution containing 0.20 m sucrose results in recovery of spermatozoon of superior qualities. In contrast to slow freezing, the ultra-rapid freezing technique of human spermatozoa seems to reduce cryoinjuries and maintain important physiological characteristics of the spermatozoa after warming.
authors.affiliation: Reproductive Medical Centre of Anhui Provincial Hospital Affiliated to Anhui Medical University, Hefei, China.
authors.forename: J
authors.initials: J
authors.lastname: Zhu
authors.forename: R-T
authors.initials: RT
authors.lastname: Jin
authors.forename: L-M
authors.initials: LM
authors.lastname: Wu
authors.forename: L
authors.initials: L
authors.lastname: Johansson
authors.forename: T-H
authors.initials: TH
authors.lastname: Guo
authors.forename: Y-S
authors.initials: YS
authors.lastname: Liu
authors.forename: X-H
authors.initials: XH
authors.lastname: Tong
mesh_terms: D000177:Acrosome / Q000502:physiology; D000328:Adult; D002462:Cell Membrane / Q000502:physiology; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004249:DNA Damage; D006801:Humans; D008297:Male; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology; D013395:Sucrose
chemical_list: D003451:Cryoprotective Agents; D013395:Sucrose
keywords: Acrosome - cryogenic vial; Cryopreservation; DNA stability; human spermatozoa - sucrose; ultra-rapid freezing